Process for reducing an alpha-keto ester

Abstract

A process for preparing (R)-hydroxy ester (II) from alpha-keto ester I comprising adding the alpha-keto ester I to a mixture comprising the ketoreductase enzyme and non-ketoreductase enzyme components, wherein the ketoreductase enzyme has a molecular weight between 36000 and 38000, and wherein the ketoreductase enzyme has an N-terminal amino acid sequence selected from the group of sequences consisting of (SEQ. ID NO. 1) Ala-Ile-Pro-Asp-Asn-Ala-Val-Leu-Glu-Gly-Ser-Leu- Val-Lys-Val-Thr-Gly-Ala-Asn-Gly (SEQ. ID NO. 4) Met-Ala-Ile-Pro-Asp-Asn-Ala-Val-Leu-Glu-Gly-Ser- Leu-Val-Lys-Val-Thr-Gly-Ala-Asn-Gly, and (SEQ. ID NO. 2) Met-Ala-Lys-Ile-Asp-Asn-Ala-Val-Leu-Pro-Glu-Gly- Ser-Leu-Val-Leu-Val-Thr-Gly-Ala-Asn-Gly.

1. A process for preparing (R)-hydroxy ester (II) 
       
         
           
           
               
               
           
         
       
       from alpha-keto ester I 
       
         
           
           
               
               
           
         
       
       comprising adding the alpha-keto ester I to a mixture comprising a ketoreductase enzyme and non-ketoreductase enzyme components, 
       wherein
 R 1  is C 1-4  alkyl; and 
 R 2  is selected from the group consisting of C 1-8  alkyl,
 C 1-8  alkyl, substituted with C 3-7  cycloalkyl, aryl, and 
 a 5- to 7-membered saturated or unsaturated heterocyclic ring, 
 
 
       wherein the ketoreductase enzyme has a molecular weight between 36000 and 38000, and wherein the ketoreductase enzyme has an N-terminal amino acid sequence selected from the group of sequences consisting of 
       
         
           
                 
               
                   (SEQ. ID NO. 1) 
                 
                 
               
                   Ala-Ale-Pro-Asp-Asn-Ala-Val-Leu-Glu-Gly-Ser-Leu- 
                 
                     
                 
                   Val-Lys-Val-Thr-Gly-Ala-Asn-Gly 
                 
                     
                 
                 
               
                   (SEQ. ID NO. 4) 
                 
                 
               
                   Met-Ala-Ile-Pro-Asp-Asn-Ala-Val-Leu-Glu-Gly-Ser- 
                 
                     
                 
                   Leu-Val-Lys-Val-Thr-Gly-Ala-Asn-Gly, and 
                 
                     
                 
                 
               
                   (SEQ ID NO. 2) 
                 
                 
               
                   Met-Ala-Lys-Ile-Asp-Asn-Ala-Val-Leu-Pro-Glu-Gly- 
                 
                     
                 
                   Ser-Leu-Val-Leu-Val-Thr-Gly-Ala-Asn-Gly. 
                 
             
                
               
            
             
                
                
                
                
               
            
             
                
               
            
             
                
                
                
                
               
            
             
                
               
            
             
                
                
                
               
            
           
         
       
     
     
       2. A process of  claim 1 , wherein the N-terminal amino acid sequence is selected from the group consisting of 
       
         
           
                 
               
                   (SEQ. ID NO. 1) 
                 
                 
               
                   Ala-Ile-Pro-Asp-Asn-Ala-Val-Leu-Glu-Gly-Ser-Leu- 
                 
                     
                 
                   Val-Lys-Val-Thr-Gly-Ala-Asn-Gly, and 
                 
                     
                 
                 
               
                   (SEQ. ID NO. 4) 
                 
                 
               
                   Met-Ala-Ile-Pro-Asp-Asn-Ala-Val-Leu-Glu-Gly-Ser- 
                 
                    Leu-Val-Lys-Val-Thr-Gly-Ala-Asn-Gly. 
                 
             
                
               
            
             
                
                
                
                
               
            
             
                
               
            
             
                
                
               
            
           
         
       
     
     
       3. A process of  claim 2 , wherein the non-ketoreductase enzyme components comprise NADP, a cofactor recycling system comprising a hydride source and a catalyst, and a buffer suitable for maintaining a pH of between about 5 and about 10. 
     
     
       4. A process of  claim 3 , wherein temperature is between about 25 and about 40° C. 
     
     
       5. A process of  claim 4 , wherein the hydride source is glucose and the catalyst is glucose dehydrogenase. 
     
     
       6. A process of  claim 5 , wherein the mixture comprises an amount of ketoreductase between about 0.1 and about 10 g/L, an amount of NADP between about 0.1 and about 10 g/L. 
     
     
       7. A process of  claims 1 , wherein R 2  is selected from the group consisting of